Radiolabeled glucans covalently linked to a radiolabel binding moiety

ABSTRACT

This invention relates to radiodiagnostic agents and reagents for preparing such agents, and also methods for producing radiolabeled radiodiagnostic agents. Specifically, the invention relates to technetium-99m ( 99m  Tc) labeled agents, methods and kits for making the agents, and methods for using the agents to image pathological sites, including sites of infection, inflammation, cancer and atherosclerosis in a mammalian body. Specifically the agents and reagents are derivatives of oligosaccharides, more specifically β-glucans.

This is a divisional of application Ser. No. 08/098,206, filed Jul. 28,1993 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to radiodiagnostic agents and reagents forpreparing such agents, and also methods for producing radiolabeledradiodiagnostic agents. Specifically, the invention relates totechnetium-99m (^(99m) Tc) labeled agents, methods and kits for makingthe agents, and methods for using the agents to image pathologicalsites, including sites of infection, inflammation, cancer andatherosclerosis in a mammalian body. Specifically the agents andreagents are derivatives of oligosaccharides, more specificallyβ-glucans.

2. Description of the Prior Art

In the field of nuclear medicine, certain pathological conditions can belocalized or the extent of such conditions determined by imaging theinternal distribution of administered radioactively-labeled tracercompounds (i.e. radiotracers or radiopharmaceuticals) that accumulatespecifically at the pathological site. This type of procedure iscommonly known as radioimaging or scintigraphic imaging. Radioimaginghas particular advantages over other methods of diagnosis in that it isessentially non-invasive, highly sensitive, highly specific, can be usedto scan the entire body and is relatively cost-effective. A variety ofradionuclides are known to be useful for radioimaging, including ⁶⁷ Ga,⁶⁸ Ga, ^(99m) Tc, ¹¹¹ In, ¹²³ I, ¹²⁵ I or ²⁰¹ Tl.

There is a clinical need to be able to determine the location and/orextent of sites of focal or localized infection. In a substantial numberof cases conventional methods of diagnosis (such as physicalexamination, x-ray, CT and ultrasonography) fail to identify such sites(e.g., an abscess). In some cases, biopsy may be resorted to, but ispreferably avoided at least until it is necessary in order to identifythe pathogen responsible for an abscess at a known location. Identifyingthe site of such "occult" infection is important because rapidlocalization of the problem is critical to effective therapeuticintervention.

An abscess may be caused by any one of many possible pathogens, so thata radiotracer specific for a particular pathogen would have limitedscope. On the other hand, infection is almost invariably accompanied byinflammation, which is a general response of the body to tissue injury.Therefore, a radiotracer specific for sites of inflammation would beexpected to be useful in localizing sites of infection caused by anypathogen.

One of the main phenomena associated with inflammation is thelocalization of leukocytes (white blood cells), including macrophages,monocytes and neutrophils, at the site of inflammation. A radiotracerspecific for leukocytes would be useful in detecting leukocytes at thesite of a localized infection.

Currently approved nuclear medicine procedures for imaging sites ofinfection use either indium-111 labeled leukocytes (¹¹¹ In-WBC) (see,e.g. Peters, 1992, J. Nucl. Med. 33: 65-67) or gallium-67 (⁶⁷ Ga)citrate (see, e.g. Ebright et al., 1982, Arch. Int. Med. 142: 246-254).

A major disadvantage of using ¹¹¹ In-labeled WBCs is that thepreparation of the radiotracer requires sterile removal of autologousblood, sterile isolation of the leukocytes from the blood, sterilelabeling of the leukocytes using conditions that do not damage the cells(since damaged WBC are taken up by the reticuloendothelial system whenre-injected) and return (re-injection) of the (now labeled) leukocytesto the patient. Furthermore, a delay of 12 to 48 hours between injectionand imaging may be required for optimal images. While ^(99m) Tc labeledleukocytes have been used to shorten this delay period (see, e.g. Vorneet al., 1989, J. Nucl. Med. 30: 1332-1336), ex-corporeal labeling isstill required. A preferred radiotracer would be one that does notrequire removal and manipulation of autologous blood components.

⁶⁷ Ga-citrate can be administered by intravenous injection. However,this compound is not specific for sites of infection or inflammation.Moreover, a delay of up to 72 hours is often required between injectionof the radiotracer and imaging. In addition, the γ-(gamma) emissionenergies of ⁶⁷ Ga are not well suited to conventional gamma cameras.

Radiolabeled monoclonal and polyclonal antibodies raised against humanleukocytes (including monocytes, neutrophils, granulocytes and others)have been developed. ^(99m) Tc labeled antigranulocyte monoclonalantibodies (see, e.g. Lind et al., 1990, J. Nucl. Med. 31: 417-473) and¹¹¹ In-labeled non-specific human immunoglobulin (see, e.g. LaMuragliaet al., 1989, J. Vasc. Surg. 10: 20-28) have been tested for thedetection of inflammation secondary to infection. ¹¹¹ In-labeled IgGshares the disadvantages of ¹¹¹ In-labeled WBC, in that 24-48 hours arerequired between injection and optimal imaging. In addition, antibodiesare difficult to produce and are associated with safety concernsregarding potential contamination with biological pathogens (e.g.retroviruses).

In addition, the effective treatment of cancer by surgery or radiationtherapy requires knowledge of the localization and extent of thedisease. A means of monitoring the progression/regression of tumorsfollowing or during any form of therapy is also highly desirable.Advances in high-resolution imaging modalities such as CT and MRI allowthe detection of many neoplasms. However certain tumors and theirmetastases are small and difficult to localize by these methods. Nuclearmedicine offers a potentially more sensitive alternative. A radiotracerthat selectively binds to or localizes to any and all cancerous tissue,sufficiently to allow easy external detection, might be considered to bethe ultimate goal of radiodiagnostic oncology.

Also, despite remarkable advances in cardiology, coronary artery diseaseremains the leading cause of death in the U.S.. The final event in thisdisease is usually fatal myocardial infarction caused by occlusivethrombosis of one or more coronary arteries usually at the site of acomplicated atherosclerotic plaque. Therefore a means, preferablynon-invasive, of determining the localization and/or extent ofatherosclerotic plaque is highly desirable as an aid to selectingappropriate patient management. One of the most notable characteristicsof atherosclerotic plaque is the accumulation of foam cells which arelipid-laden macrophages.

β-Glucans are oligoglucosides, which comprise 1,3 and 1,6 linkedβ-D-glucose residues, originally discovered as components of yeast andfungal cell walls (Bartnicki-Garcia in Ann Rev Microbiol. 1968, 22, 87).Originally obtained in an insoluble form, β-glucans have since beenobtained as soluble, low molecular weight oligomers (Janusz, Austen andCzop, J. Immunol. (1989), 142, (959-965). They have been shown to beactive in enhancing the host defense mechanisms of mammals by activatingthe alternative complement pathway through their specific binding toreceptors (called β-glucan receptors) found on the cell-surfaces ofmonocytes, macrophages and neutrophils (Czop and Kay, J. Exp. Med.(1991), 173, 1511-1520, Czop et al, Biochemistry of the Acute AllergicReactions: Fifth International Symposium. (1989), 287-296 and J. K.Czop, Pathol. Immunopathol. Res (1986), 5, 286-296, Czop and Austen, J.Immunol. (1985), 134, 2588-2593). The in vivo administration ofparticulate β-glucans has been shown to provide protection from manypathogens including bacteria, viruses and fungi as well as reducingtumor growth (Czop et al, Biochemistry of the Acute Allergic Reactions:Fifth International Symposium. 1989, 287-296). The smallest activeβ-glucan reported so far is a heptaglucoside (Janusz et al, J Immunol1989, 142, 959. Onderdonk and co-workers (Infection and Immunity, 1992,60, 1642-1647) describe the antiinfective properties of this smallβ-glucan. The β-glucans have also been shown to exhibit an anti-tumorgrowth effect, believed to occur by increasing the number of macrophageslocalizing to tumors (Di Luzio in Pathophysiology of theReticuloendothelial System (Eds Altruo and Saba), Raven Press, NY,pp209-224).

Czop and Janusz, U.S. Pat. No. 5,057,503 (1991), claim a heptaglucosidecapable of reacting with β-glucan receptors, their isolation and theirtherapeutic use.

Jamas et al, PCT/US90/03440 claim β-glucans as drug delivery vehiclesand as adjuvants.

Jamas et al, PCT/US90/05022 claim a method of activating the immunesystem by administering β-glucans.

Jamas et al, PCT/US90/05041 claim a method of producing a solubleβ-glucan.

Methods for preparing radiolabel-binding moieties and of labeling themwith ^(99m) Tc are disclosed in co-pending U.S. patent applications Ser.Nos. 07/653,012, now abandoned, which issued as U.S. Pat. No. 5,654,272;07/757,470, now U.S. Pat. No. 5,225,180; 07/807,062, now U.S. Pat. No.5,443,815; 07/851,074, now abandoned, which issued as U.S. Pat. No.5,711,931; 07/871,282, a divisional of which issued as U.S. Pat. No.5,720,934; 07/886,752, now abandoned, a continuation of which has beenallowed as USSN 08/273,274; 07/893,981, now U.S. Pat. No. 5,508,020;07/955,466; 07/977,628, now U.S. Pat. No. 5,405,597; 08/019,525, nowU.S. Pat. No. 5,552,525; 08/044,825, now abandoned, which issued as U.S.Pat. No. 5,645,815; and 08/073,577, now U.S. Pat. No. 5,561,220; and PCTInternational Applications PCT/US92/00757, PCT/US92/10716,PCT/US93/02320, PCT/US93/03687, PCT/US93/04794, and PCT/US93/06029,which are hereby incorporated by reference.

SUMMARY OF THE INVENTION

The present invention provides scintigraphic imaging agents that areβ-glucans which are radiolabeled with a radioisotope or areβ-glucan-derived reagents radioactively-labeled with a radioisotope. Theβ-glucan-derived reagents of the invention are comprised of a β-glucancovalently linked to a radiolabel binding moiety. The scintigraphicimaging agents of this invention are useful for imaging pathologicalsites within a mammalian body including sites of infection,inflammation, cancer and atherosclerosis.

A first aspect of the invention comprises reagents for preparingscintigraphic imaging agents for imaging sites within a mammalian body,said reagents comprising a β-glucan having a 1,3 and 1,6 linkedD-glucoside sequence, of molecular weight of up to about 2,000 kDa and aradiolabel-binding moiety.

In a second aspect, the scintigraphic imaging agent of the inventioncomprises a soluble β-glucan.

In a third aspect, the scintigraphic imaging agent of the inventioncomprises the radioisotope ^(99m) Tc.

In another aspect of the invention the radiolabel-binding moiety islinked to the β-glucan via a 1-amino or 1-thio substituent.

In yet another aspect, the reagents of the invention comprise a β-glucanand a radiolabel-binding moiety of formula

I

    Cp(aa)Cp

wherein Cp is a protected cysteine residue and (aa) stands for an aminoacid, and wherein the radiolabel-binding moiety is covalently linked tothe β-glucan. In a preferred embodiment, the amino acid is glycine. Inanother preferred embodiment, the radiolabel-binding moiety is linked tothe β-glucan via a linker which forms either an ether, thioether oramine bond to the β-glucan.

In another aspect, the invention provides reagents comprising aradiolabel-binding moiety having the following structure: radioisotopecomplexing group comprising a single thiol moiety having the followingstructure

II

    A-CZ(B)- C(R.sup.1 R.sup.2)!.sub.n -X

wherein A is H, HOOC, H₂ NOC, (β-glucan)-(linker)-NHOC,(β-glucan)-(linker)-OOC or R⁴ ; B is H, SH or --NHR³,--N(R³)-(linker)-(β-glucan) or R⁴ ; X is SH or --NHR³,--N(R³)-(linker)-(β-glucan) or R⁴ ; R¹, R², R³ and R⁴ are independentlyH or straight or branched chain or cyclic lower alkyl; n is 0, 1 or 2;and: (1) where B is --NHR³ or --N(R³)-(linker)-(β-glucan), X is SH and nis 1 or 2; (2) where X is --NHR³ or --N(R³)-(linker)-(β-glucan), B is SHand n is 1 or 2; (3) where B is H or R⁴, A is HOOC, H₂ NOC,(β-glucan)-(linker)-NHOC or (β-glucan)-(linker)-OOC, X is SH and n is 0or 1; (4) where A is H or R⁴, then where B is SH, X is --NHR³ or--N(R³)-(linker)-(β-glucan) and where X is SH, B is --NHR³ or--N(R³)-(linker)-(β-glucan); (5) where X is H or R⁴, A is HOOC, H₂ NOC,(β-glucan)-(linker)-NHOC or (β-glucan)-(linker)-OOC and B is SH; (6)where Z is methyl, X is methyl, A is HOOC, H₂ NOC,(β-glucan)-(linker)-NHOC or (β-glucan)-(linker)-OOC and B is SH and n is0; and wherein the thiol moiety is in the reduced form.

In yet another aspect, the present invention provides reagentscomprising β-glucans covalently linked to a radiolabel-binding moietyhaving the following structure: ##STR1## For purposes of this invention,radiolabel-binding moieties having structure III will be referred to aspicolinic acid (Pic)-based moieties; or ##STR2## For purposes of thisinvention, radiolabel-binding moieties having structure IV will bereferred to as picolylamine (Pica)-based moieties; wherein X is H or aprotecting group; (amino acid) is any amino acid. In a preferredembodiment, the amino acid is glycine and X is an acetamidomethylprotecting group.

In yet another embodiment of the invention, reagents are provided forpreparing scintigraphic imaging agents for imaging sites within amammalian body, comprising a β-glucan and a bisamino bisthiolradiolabel-binding moiety covalently linked to the β-glucan. Thebisamino bisthiol radiolabel-binding moiety in this embodiment of theinvention has a formula selected from the group consisting of: ##STR3##wherein each R⁵ can be independently H, CH₃ or C₂ H₅ ; each (pgp)⁵ canbe independently a thiol protecting group or H; m, n and p areindependently 2 or 3; A is linear or cyclic lower alkyl, aryl,heterocyclyl, combinations or substituted derivatives thereof; and X is(linker)-β-glucan; ##STR4## wherein each R⁵ is independently H, loweralkyl having 1 to 6 carbon atoms, phenyl, or phenyl substituted withlower alkyl or lower alkoxy; m, n and p are independently 1 or 2; A islinear or cyclic lower alkyl, aryl, heterocyclyl, combinations orsubstituted derivatives thereof; V is H or --CO-(linker)-β-glucan; R⁶ isH or a (linker)-β-glucan; provided that when V is H, R⁶ is a(linker)-β-glucan and when R⁶ is H, V is a --CO-(linker)-β-glucan. Forpurposes of this invention, radiolabel-binding moieties having thesestructures will be referred to as "BAT" moieties.

The invention comprises scintigraphic imaging agents that are complexesbetween β-glucans or the reagents of the invention and ^(99m)

Tc, and methods for radiolabeling the β-glucans and reagents of theinvention with ^(99m) Tc. Radiolabeled complexes provided by theinvention may be formed by reacting β-glucans or the reagents of theinvention with ^(99m) Tc in the presence of a reducing agent. Preferredreducing agents include but are not limited to dithionite ion, stannousion and ferrous ion. Complexes of the invention are also formed bylabeling β-glucans or the reagents of the invention with ^(99m) Tc byligand exchange of a prereduced ^(99m) Tc complex as provided herein.

The invention also provides kits for preparing scintigraphic imagingagents that are β-glucans or the reagents of the invention radiolabeledwith ^(99m) Tc. Kits for labeling the β-glucans or the reagents providedby the invention with ^(99m) Tc are comprised of a sealed vialcontaining a predetermined quantity of a β-glucan or a reagent of theinvention and a sufficient amount of reducing agent to label theβ-glucan or reagent with ^(99m) Tc.

This invention provides methods for using scintigraphic imaging agentsthat are radiolabeled β-glucans and reagents for imaging pathologicalsites, including infection, inflammation, cancer and atherosclerosiswithin a mammalian body by obtaining in vivo gamma scintigraphic images.These methods comprise administering an effective diagnostic amount ofradiolabeled β-glucan or reagent of the invention and detecting thegamma radiation emitted by the radiolabel localized at the pathologicalsite within the mammalian body.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

DETAILED DESCRIPTION OF THE INVENTION

The β-glucans of this invention have linear or branched 1,3 and 1,6linked D-glucoside sequences. They comprise both insoluble and solublemolecular entities having molecular weights of up to about 2,000 kDa. Ina preferred embodiment, the β-glucan is soluble. Most preferably thesoluble β-glucan is a poly-β1-6-glucotriosyl-β1-3-glucopyranose.

In Cp(aa)Cp-containing β-glucan reagents, the Cp is a protected cysteinewhere the s-protecting groups are the same or different and may be butare not limited to:

--CH₂ -aryl (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--CH-(aryl)₂, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--C-(aryl)₃, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--CH₂ --(4-methoxyphenyl);

--CH-(4-pyridyl)(phenyl)₂ ;

--C(CH₃)₃

-9-phenylfluorenyl;

--CH₂ NHCOR (R is unsubstituted or substituted alkyl or aryl);

--CH₂ --NHCOOR (R is unsubstituted or substituted alkyl or aryl);

--CONHR (R is unsubstituted or substituted alkyl or aryl);

--CH₂ --S--CH₂ -phenyl

The preferred protecting group has the formula --CH₂ --NHCOR wherein Ris a lower alkyl having 1 and 8 carbon atoms, phenyl orphenyl-substituted with lower alkyl, hydroxyl, lower alkoxy, carboxy, orlower alkoxycarbonyl.

β-Glucans of the present invention can be obtained from natural sources,such as yeast, by methods well known in the art: (e.g. see Manners,Masson and Patterson in J. Gen. Microbiol. (1974), 80, 411-417). Smallsoluble β-glucans can be obtained from larger β-glucans by methods knownin the art (e.g. as described by Janusz, Austen and Czop in J. Immunol.(1989),142, 959-965 and Jamas et al, PCT/US90/05041) or can be obtainedby chemical synthesis. Preferred soluble β-glucans arepoly-β1-6-glucotriosyl-β1-3-glucopyranoses including those that areheptaglucosides. The term soluble β-glucan is used herein to meansoluble in a physiologically compatible solution to about 10 mg/mL.

The reagents of this invention comprise a β-glucan covalently attachedto a radiolabel-binding moiety. The radiolabel binding moiety can beattached directly to the β-glucan or it can be attached via a linker.The direct attachment of the radiolabel-binding moiety may beadvantageously made by a 1-thioether or 1-amino group, or via an esteror ether bond to any hydroxyl group of the β-glucan (see for example,Her, Santikarn and Reinhold, J. Carbohydrate Chemistry (1987), 6,129-139 and Bogwald, Seljelid and Hoffman, Carbohydrate research (1986),148, 101-107). The linker is normally a small entity, of less than about500 Da formula weight and may advantageously be a small ( up to about 10carbon atoms) linear or branched chain divalent alkyl, alkaryl or arylgroup, optionally comprising a multiplicity of hetero atoms, preferablyoxygens, and optionally substituted, preferably with hydrophilicmoieties.

In forming a complex of radioactive technetium with the β-glucans andthe reagents of this invention, the technetium complex, preferably asalt of ^(99m) Tc pertechnetate, is reacted with the β-glucan or reagentin the presence of a reducing agent. Preferred reducing agents aredithionite, stannous and ferrous ions; the most preferred reducing agentis stannous chloride. Means for preparing such complexes areconveniently provided in a kit form comprising a sealed vial containinga predetermined quantity of a β-glucan or reagent of the invention to belabeled and a sufficient amount of reducing agent to label the reagentwith ^(99m) Tc. Alternatively, the complex may be formed by reacting aβ-glucan or reagent of this invention with a pre-formed labile complexof technetium and another compound known as a transfer ligand. Thisprocess is known as ligand exchange and is well known to those skilledin the art. The labile complex may be formed using such transfer ligandsas tartrate, citrate, gluconate or mannitol, for example. Among the^(99m) Tc pertechnetate salts useful with the present invention areincluded the alkali metal salts such as the sodium salt, or ammoniumsalts or lower alkyl ammonium salts.

The reaction of β-glucans and reagents of this invention withTc-pertechnetate or preformed ^(99m) Tc labile complex can be carriedout in an aqueous medium at room temperature or with heating for a shortperiod (from 5 to about 60 minutes). When an anionic complex having acharge of -1! is formed in the aqueous medium in the form of a salt witha suitable cation such as sodium cation, ammonium cation, mono, di- ortri-lower alkyl amine cation, etc. Any conventional salt of the anioniccomplex with a pharmaceutically acceptable cation can be used inaccordance with this invention.

In a preferred embodiment of the invention, a kit for preparing ^(99m)Tc-labeled β-glucans and β-glucan reagents is provided. An appropriateamount of the β-glucan or reagent is introduced into a vial containing areducing agent, such as stannous chloride, in an amount sufficient tolabel the β-glucan or reagent with ^(99m) Tc. An appropriate amount of atransfer ligand as described (such as tartrate, citrate, gluconate ormannitol, for example) can also be included. In forming the ^(99m) Tccomplexes, it is generally preferred to form radioactive complexes insolutions containing radioactivity at concentrations of from about 0.01millicurie (mCi) to 100 mCi per ml.

Scintigraphic imaging agents of this invention can also be prepared byincubating radiolabeled β-glucans or radiolabeled β-glucan reagents withleukocytes, wherein the leukocytes take up the radiolabeled species andcan then be administered as radiolabeled leukocytes.

The radiolabeled scintigraphic imaging agents provided by the presentinvention can be used for visualizing pathological sites including sitesof inflammation and infection, including abscesses and sites of "occult"infection and inflammatory bowel disease. The imaging agents providedcan also be used to image sites of atherosclerotic plaque and alsotumors. In accordance with this invention, the scintigraphic imagingagents are administered in a single unit injectable dose. Any of thecommon carriers known to those with skill in the art, such as sterilesaline solution or plasma, can be utilized after radiolabeling forpreparing the injectable solution to diagnostically image variousorgans, tumors and the like in accordance with this invention.Generally, the unit dose to be administered has a radioactivity of about0.01 mCi to about 100 mCi, preferably 1 mCi to 20 mCi. The solution tobe injected at unit dosage is from about 0.01 ml to about 10 ml. Afterintravenous administration, imaging of the organ or tumor in vivo cantake place in a matter of a few minutes. However, imaging can takeplace, if desired, in hours or even longer, after injecting intopatients. In most instances, a sufficient amount of the administereddose will accumulate in the area to be imaged within about 0.1 of anhour to permit the taking of scintiphotos. Any conventional method ofscintigraphic imaging for diagnostic purposes can be utilized inaccordance with this invention.

The scintigraphic imaging agents provided by the invention may beadministered intravenously in any conventional medium for intravenousinjection such as an aqueous saline medium, or in blood plasma medium.Such medium may also contain conventional pharmaceutical adjunctmaterials such as, for example, pharmaceutically acceptable salts toadjust the osmotic pressure, buffers, preservatives and the like. Amongthe preferred media are normal saline and plasma.

The methods for making and labeling these compounds are more fullyillustrated in the following Examples. These Examples illustrate certainaspects of the above-described method and advantageous results. TheseExamples are shown by way of illustration and not by way of limitation.

EXAMPLE 1 Reagent Synthesis

DMSO=dimethyl sulfoxide, DMF=N,N-dimethylformamide andDIEA=N,N-diisopropylethylamine.

Poly-β1-6-glucotriosyl-β1-3-glucopyranose (PGG) is obtained using theprocedures described by Jamas et al (PCT/US90/05041).

N-α-Boc-lysyl-glycyl-(S-trityl)cysteine amide,glycyl-glycyl-(S-trityl)cysteine amide andchloroacetyl-(S,S'-bis-acetamidomethyl)cysteinyl-glycyl-cysteine amideare prepared by solid phase or solution phase peptide synthesis and arepurified by reverse phase HPLC.

A conjugate with N¹,N⁴-bis(2-mercapto-2-methylpropyl)-1,4,10-triazadecane is obtained byreacting a β-glucan (e.g., PGG) at from about 1 to 100 mg/mL with about1.5 mmol N¹ -(t-butoxycarbonyl) -N¹,N⁴-bis(2-methyl-2-triphenylmethylthiopropyl)-1,4,10-triazadecane in water,Cellosolve or mixtures thereof at approximately pH 7 at about 65° C. forfrom 1 to about 10 hours, followed by reduction with NaBH₃ CN followedby deprotection with trifluoroacetic acid. The product is purified bypreparative HPLC.

Similarly conjugates of ε-(lysyl-glycyl-cysteine amide) andglycyl-glycyl-cysteine amide are prepared fromN-α-Boc-lysyl-glycyl-(S-trityl)cysteine amide andglycyl-glycyl-(S-trityl)cysteine amide respectively.

A conjugate of N⁶,N⁹ -bis(2-mercapto-2-methylpropyl)-6,9-diazanonanoicacid is prepared by reacting β-glucan (e.g. PGG) at from about 1 to 100mg/mL in water, DMSO or DMF containing about 1.5 mmol DIEA andoptionally containing about 0.15 mmol 4-dimethylaminopyridine, withabout 1.5 mmol of the N-hydroxysuccinimide ester of N⁹-(t-butoxycarbonyl)-N⁶,N⁹-bis(2-methyl-2-triphenylmethylthiopropyl)-6,9-diazanonanoic acid, atroom temperature, followed by deprotection with TPA and purification byHPLC.

A conjugate of (S,S'-bis-acetamidomethyl)cysteinyl-glycyl-cysteine amideis prepared by reacting β-glucan (e.g. PGG) at from about 1 to 100 mg/mLin DMSO, with sodium methylsulfinylmethanide, or another suitable base,(approx. 1.6 mmol base/100 mg β-glucan) for from 1 to about 24 hours andreacting the resultant mixture with approx. 1.6 mmolchloroacetyl-(S,S'-bis-acetamidomethyl)cysteinyl-glycyl-cysteine amidefor about 1 to 5 hours at between 20° and 50° C., followed bypurification by HPLC.

EXAMPLE 2 A General Method for Radiolabeling with Tc-99m

1. About 0.1 mg of a β-glucan or a reagent prepared as in Example 1 isdissolved in 0.1 mL of water or 50/50 ethanol/water. Approximately 100μg stannous salt as stannous chloride pre-dissolved in methanol, orstannous tartrate pre-dissolved in water is added followed by 1-10 mCi^(99m) Tc pertechnetate in approximately 0.1 mL. The mixture is allowedto stand for 15-30 minutes at room temperature or at 100° C. For solubleβ-glucans the preparation is then filtered through a 0.2 μm filter andthe Tc-99m labeled product purity is determined by HPLC. The purity ofinsoluble β-glucan products is assessed by ITLC developed in saline.

2. About 0.1 mg of β-glucan or reagent prepared as described in Example1 is dissolved in 0.1 mL of water or 50/50 ethanol/water orphosphate-buffered saline or 50 mM potassium phosphate buffer (pH=5, 6or 7.4). Tc-99m gluceptate was prepared by reconstituting a Glucoscanvial (E.I. DuPont de Nemours, Inc.) with 1.0 mL of Tc-99m sodiumpertechnetate containing up to 200 mCi and allowed to stand for 15minutes at room temperature. 25 μl of Tc-99m gluceptate was then addedto the peptide and the reaction allowed to proceed at room temperatureor at 100° C. for 15-30 min. For soluble β-glucans the preparation isthen filtered through a 0.2 μm filter and the Tc-99m labeled productpurity is determined by HPLC. The purity of insoluble β-glucan productsis assessed by ITLC developed in saline.

What is claimed is:
 1. A composition comprising a reagent comprising aspecific binding β-glucan covalently linked to a radiometal-bindingmoiety, wherein the β-glucan is linked to the moiety by a linkageselected from the group consisting of a direct covalent linkage and alinker having a molecular weight less than about 500 Da; and a stannousion.
 2. A kit for preparing a radiopharmaceutical preparation, said kitcomprising a sealed vial containing:a) a predetermined quantity of areagent comprising specific binding β-glucan covalently linked to aradiometal-binding moiety , wherein the β-glucan is linked to the moietyby a linkage selected from the group consisting of a direct covalentlinkage and a linker having a molecular weight less than about 500 Da;and b) a sufficient amount of reducing agent to label the reagent with^(99m) Tc.
 3. The composition of claim 1, wherein the β-glucan issoluble in a physiologically compatible solution to about 10 mg/ml.
 4. Acomposition comprising a poly-β1-6-glucotriosyl-β1-3-glucopyranosecovalently linked to a radiometal-binding moiety, and a stannous ion. 5.A composition comprising a stannous ion and a specific binding β-glucancovalently linked to a radiometal-binding moiety having a formulaselected from the group consisting of:(a) C(pgp)^(s) -(aa)-C(pgp)^(s)wherein (pgp)^(s) is H or a thiol protecting group and (aa) is anyprimary α- or β-amino acid; (b) a radiometal complexing group comprisinga single thiol moiety having a formula:

    A-CZ(B)- C(R'R")!.sub.n -X

wherein A is H, HOOC, H₂ NOC, (β-glucan)-(linker)-NHOC,(β-glucan)-(linker)-OOC or R""; B is H, SH, --NHR'",--N(R'")-(linker)-(β-glucan), or R""; X is H, SH, --NHR'",--N(R'")-(linker)-(β-glucan) or R""; Z is H or R""; R', R", R'" and R""are independently H or lower straight or branched chain or cyclic alkyl;n is 0, 1 or2; and where B is --NHR'" or --N(R'")-(linker)-(β-glucan), Xis SH, and n is 1 or 2; where X is --NHR'" or--N(R'")-(linker)-(β-glucan), B is SH, and n is 1 or 2; where B is H orR"", A is HOOC, H₂ NOC, (β-glucan)-(linker)-NHOC,(β-glucan)-(linker)-OOC, X is SH, and n is 0 or 1; where A is H or R"",then where B is SH, X is --NHR'" or --N(R'")-(linker)-(β-glucan) andwhere X is SH, B is --NHR'" or --N(R'")-(linker)-(β-glucan); where X isH or R"", A is HOOC, H₂ NOC, (β-glucan)-(linker)-NHOC,(β-glucan)-(linker)-OOC and B is SH; where Z is methyl, X is methyl, Ais HOOC, H₂ NOC, (β-glucan)-(linker)-NHOC, (β-glucan)-(linker)-OOC, B isSH and n is 0; and wherein (β-glucan) represents the covalent attachmentof the radiometal binding moiety to the specific binding β-glucan;##STR5## wherein X=H or a protecting group; (amino acid)=any amino acid;##STR6## wherein X=H or a protecting group; (amino acid)=any amino acid;##STR7## wherein each R is independently H, CH₃ or C₂ H₅ ; each(pgp)^(s) is independently a thiol protecting group or H; m, n and p areindependently 2 or 3; A =linear or cyclic lower alkyl, aryl,heterocyclyl, a combination thereof or a substituted derivative thereof;##STR8## wherein each R is independently H, CH₃ or C₂ H₅ ; m, n and pare independently 2 or 3; A=linear or cyclic lower alkyl, aryl,heterocyclyl, a combination thereof or a substituted derivative thereof;V=H or --CO-(linker)-(β-glucan); R'=H or (linker)-(β-glucan); andwherein when V=H, R'=-(linker)-(β-glucan) and when R'=H,V=--CO-(linker)-(β-glucan); wherein (linker) is a bond or a divalentradical covalently attached to the β-glucan and to theradiolabel-binding moiety and wherein (β-glucan) represents the covalentattachment of the radiometal binding moiety to the β-glucan.
 6. The kitof claim 2, wherein the β-glucan is soluble in a physiologicallycompatible solution to about 10 mg/ml.
 7. A kit comprising a sealed vialcontaining:a) a predetermined quantity of a reagent comprising apoly-β1-6-glucotriosyl-β1-3-glucopyranose covalently linked to aradiolabel-binding moiety: and b) a sufficient amount of a reducingagent to label the reagent with ^(99m) Tc.
 8. A kit comprising a sealedvial containing:a) a predetermined quantity of a reagent comprising aspecific binding β-glucan covalently linked to a radiometal-bindingmoiety having a formula selected from the group consisting of: (a)C(pgp)^(s) -(aa)-C(pgp)^(s) wherein (pgp)^(s) is H or a thiol protectinggroup and (aa) is any primary α- or β-amino acid; (b) a radiometalcomplexing group comprising a single thiol moiety having a formula:

    A-CZ(B)- C(R'R")!.sub.n -X

wherein A is H, HOOC, H₂ NOC, (β-glucan)-(linker)-NHOC,(β-glucan)-(linker)-OOC or R""; B is H, SH, --NHR'",--N(R'")-(linker)-(β-glucan), or R""; X is H, SH, --NHR'",--N(R'")-(linker)-(β-glucan) or R""; Z is H or R""; R', R", R'" and R""are independently H or lower straight or branched chain or cyclic alkyl;n is 0, 1 or2; and where B is --NHR'" or --N(R'")-(linker)-(β-glucan), Xis SH, and n is 1 or 2; where X is --NHR'" or--N(R'")-(linker)-(β-glucan), B is SH, and n is 1 or 2; where B is H orR"", A is HOOC, H₂ NOC, (β-glucan)-(linker)-NHOC,(β-glucan)-(linker)-OOC, X is SH, and n is 0 or 1; where A is H or R"",then where B is SH, X is --NHR'" or --N(R'")-(linker)-(β-glucan) andwhere X is SH, B is --NHR'" or --N(R'")-(linker)-(β-glucan); where X isH or R"", A is HOOC, H₂ NOC, (β-glucan)-(linker)-NHOC,(β-glucan)-(linker)-OOC and B is SH; where Z is methyl, X is methyl, Ais HOOC, H₂ NOC, (β-glucan)-(linker)-NHOC, (β-glucan)-(linker)-OOC, B isSH and n is 0; and wherein (β-glucan) represents the covalent attachmentof the radiometal binding moiety to the specific binding β-glucan;##STR9## wherein X=H or a protecting group; (amino acid)=any amino acid;##STR10## wherein X =H or a protecting group; (amino acid)=any aminoacid; ##STR11## wherein each R is independently H, CH₃ or C₂ H₅ ; each(pgp)^(s) is independently a thiol protecting group or H; m, n and p areindependently 2 or 3; A=linear or cyclic lower alkyl, aryl,heterocyclyl, a combination thereof, or a substituted derivativethereof; ##STR12## wherein each R is independently H, CH₃ or C₂ H₅ ; m,n and p are independently 2 or 3; A=linear or cyclic lower alkyl, aryl,heterocyclyl, a combination thereof, or a substituted derivativethereof; V=H or --CO-(linker)-(β-glucan); R'=H or (linker)-(β-glucan);and wherein when V=H, R'=-(linker)-(β-glucan) and when R'=H,V=--CO-(linker)-(β-glucan); wherein (linker) is a bond or a divalentradical covalently attached to the β-glucan and to theradiolabel-binding moiety and wherein (β-glucan) represents the covalentattachment of the radiometal binding moiety to the specific bindingβ-glucan; and b) a sufficient amount of a reducing agent to label thereagent with ^(99m) Tc.
 9. A composition comprising reagent comprising aβ-glucan comprising a multiplicity of 1,3- and 1,6-linked β-glucosideresidues covalently linked to a radiometal-binding moiety.
 10. A kitcomprising a reagent comprising a β-glucan comprising a multiplicity of1,3- and 1,6-linked β-glucoside residues covalently linked to aradiometal-binding moiety.